The retrovirus HIV (Human Immunodeficiency Virus) is the causative agent of acquired immune deficiency syndrome (AIDS). Replication of HIV has in common with other retroviruses and lytic RNA viruses, most notably the picornaviruses, the synthesis of polyprotein precursors which are subsequently processed to yield mature viral products. It is now known that the proteolytic enzyme involved in the formation of core proteins during replication of HIV encoded within the viral genome, and by analogy with other retroviruses and the picornaviruses, it may be assumed that there are no homologous enzymes present in the uninfected cell. The specific aim of this project is to develop an in vitro system in which the proteolysis of the gag gene precursor can be studied, and to develop specific inhibitors of the protease that may be useful as therapeutic agents blocking virus spread. We will study in vitro the synthesis of the HIV-specific protease and its activity on a natural substrate. The amino terminal coding region of the pol gene of HIV will be inserted into suitable plasmids containing cloning sites behind a promoter for bacteriophage T7 RNA polymerase. The T7 polymerase operates very rapidly and nonspecifically on such engineered plasmids to produce large quantities of RNA, which will be isolated and used to direct synthesis of protease in reticulocyte lysates. The proteolytic activity of the translation product will be tested by assaying for processing of the gag precursor supplied in trans into proteins of the same size as those in virions. The gag precursor will be synthesized independently in reticulocyte lysates, using the RNA obtained by transcription of the gag gene of HIV cloned behind T7 promoters. We will define by mutation the minimum substrate primary sequence required for processing, the role of substrate conformation as a determinant of cleavage, whether post-translational modifications of the substrate (phosphorylation and myristylation) occur in vitro, and the role of these modifications in efficient processing. Synthetic protease inhibitors directed against sequence and conformational targets will be developed and tested for antiviral specificity in vitro. Drugs identified in this study potentially may be clinically- significant inhibitors of virus spread.